The regulated synthesis of mRNA plays a central role in nearly all critical biological processes in animals, from development to the immune response to memory. Great progress has been made over the last few decades in understanding many of the basic biochemical mechanisms of transcriptional regulation and in identifying and dissecting a limited number of cis-sequences that control when and where specific genes are expressed. Even with these advances and despite the importance of gene regulation in the basic biology of all organisms, our ability our ability to annotate regulatory information in animal genomes is extremely limited. The availability of genome sequences from an increasing number of animals including humans, coupled with systematic experimental characterizations of animal transcriptional regulatory networks, offers a great opportunity to better understand how transcriptional regulatory information is encoded in genome sequences and to develop tools that utilize this knowledge. Although the number of well-characterized animal cis-regulatory regions is small, there is an emerging view that there are common design principles that govern the organization and activities of these sequences. Here, we propose to take advantage of the increasing amount of genomic data to develop strategies and computational tools to better understand and exploit these design principles to characterize the transcriptional regulatory content of animal genomes. Using the Drosophila embryo as a model system, we will integrate analyses of the publicly available genome sequences of D. melanogaster and D. pseudoobscura, in situ gene expression patterns of all 13,700 Drosophila genes, and the experimentally determined binding specificities of all 100 transcription factors active in the early Drosophila embryo to: a. Identify sequences in the Drosophila genome that control transcription in the embryo and relate their sequences to the specific patterns of expression they direct. b. Better understand the rules that govern the organization and activity of cis-regulatory sequences. c. Develop methods to infer significant features of animal cis-regulatory networks from limited experimental data. We will maintain a website to provide tools for the analysis of cis-sequences to the Drosophila community, and will make all of these tools freely available for those studying other organisms. Although we will focus our efforts on the Drosophila embryo, we believe the approaches and tools we develop will be of general use in the annotation of animal genomes.